The research was carried out in the Department of Physiology of the Nelson R Mandela School of Medicine under the supervision of Professor Maurice Mars. This study was conducted with the approval of the Ethics Committee of the University of Natal. Lymphocyte apoptosis was again demonstrated after exercise, however the percentage of apoptosis was a maximum of 4.8% at 24 hours.
The relationship between exercise intensity and the risk of upper respiratory infections has been modeled in the form of a J-shaped curve (Niemann, 1994). Following this, the cells experience an irreversible condensation of the cytoplasm, accompanied by an increase in cell density, dense aggregation of cytoplasmic organelles, and condensation of the nuclear chromatin to form dense granular caps underlying the nuclear membrane. Another characteristic feature of apoptosis is that the dying cells disappear rapidly from the tissue without generating any inflammatory reaction.
Changes associated with a cell undergoing necrotic death are cell swelling, cytosolic as well as nuclear structural changes, and preservation of euchromatin and nuclear pores. During the last years the field of apoptosis has found application in various fields and ahs have been reported in several thousands of scientific publications. Recent advances have revealed that mitochondria probably play a central regulatory role in the field of apoptosis, particularly through the cytochrome c pathway.
These intracellular changes are sufficient to induce the formation of mitochondrial permeability transition pores, leading to the subsequent release of cytochrome c and the activation of the caspase cascade.
Introduction
THE COMET ASSAY
- Blood preparation and treatments
- Slide Preparation
- Electrophoresis
- Staining
- Image analysis
- Statistical analysis
- Results
- Introduction
The detection of DNA breaks is facilitated by alkaline electrophoresis of cells embedded in agarose and lysis by detergents with a high salt concentration (Vijayalaxmi et al., 1992). Breaks in DNA strands migrate toward the anode, producing an image that resembles a comet. The sensitivity of the comet assay in the evaluation of DNA damage depends on accurate and reproducible measurement of DNA in the comet head and tail regions (Olive et al., 1992).
The purpose of this study was to assess whether a single period of high intensity. The procedure described for SCGE analysis by Singh et al., (1988) was followed with minor modifications: 200 microliters of 0.75% agarose diluted in Ca++ and Mg++. The slides were immediately covered with a coverslip and kept in the refrigerator for another 5 minutes to solidify the LMP A.
The coverslips were again removed and a final top coat of 75 µl of 0.5% LMPA at 37°C was added and re-chilled for a further 5 minutes. The coverslips were then carefully removed and the slides were immersed in a container containing cold lysing solution (2.5 M NaCl, 100 mM EDT A, 1% Triton The tank was carefully filled with freshly made alkaline buffer (300 mM NaOH and ImM EDTA, pH 13.0) to a level of approximately 0.30 mm above the slides.
Slides were allowed to stand in electrophoresis buffer for 20 min to allow DNA to degrade prior to electrophoresis. Slides were allowed to stand in Tris for 5 min and this step was repeated three times. Finally, the slides were stained by placing 40 µl of 20 µg/ml ethidium bromide solution on each slide and then covering them with a cover slip.
Data were presented as the mean tail moment for the cells plus or minus the standard deviation within the different time intervals. The mean tail moments, an indication of the severity of the DNA damage, are shown in table 1 and figure 4. Post hoc testing showed significant increases in mean tail moments, 24 and 48 hours after testing (** = .. p However, electron light and ultraviolet microscopy remain the gold standard in the analysis of apoptosis, although these techniques are rather tedious (Gorczyca et al., 2000). These methods are all designed around the changes in size and gross cell structure, in plasma membrane transport function, physical integrity or in chromatin and DNA structure (Gorczyca et ai., 2000). Timely generation of recognition signals on the surface of apoptotic cells is therefore a key event in the apoptotic program (Verhoeven et al., 1995). Phospholipids found in the plasma membrane are asymmetrically distributed between the inner and outer leaflets of the plasma membrane. After spinning, the supernatant was decanted and the pellet was resuspended in the remaining liquid. The staining solution was prepared by adding 20)/l of both annexin V-FITC and propidium iodide to 1000)/l incubation buffer. The cells were then treated with 100 ml of staining solution and incubated for 15 minutes at 22°C to allow the cells to take up the respective stains. The cells were then analyzed on a Becton Dickenson Facs Calibur flow cytometer using a 488 nm excitation and 515 nm bandpass filter for fluorescence detection and a filter larger than 560 nm for propidium iodide detection. Electronic compensation of the instrument was performed to eliminate overlap of the two emission spectra. The results of the annexin V assays showing the percentage of cells found to be apoptotic or necrotic at the different time points are shown in Table 2. Post hoc testing using the Tukey Kramer multiple comparisons test showed that the tests after 48 hours were significantly lower than all other time points for both percentage of apoptosis and. Measurement of oxygen consumption in athletes was measured with computerized open spirometry (Oxycon Gamma Spirometer). The results therefore show that there was no DNA migration before exercise, while after running most cells showed increased DNA migration. This therefore rules out the possibility that the cytotoxic effects in the SCGE assay are due to acidic conditions (Hartmann et al., 1994). Therefore apoptosis due to vigorous exercise would be easily detected by the SCGE technique which is sensitive in detecting DNA single-strand breaks. As expected once again, as in the SCGE analysis, there is a large degree of variability between test subjects. Three of the test subjects showed a marked increase in apoptosis levels after 24 hours, while the other two remained about the same. In Nieman's "J-shaped model", it is suggested that exercise can increase or decrease immunity depending on the frequency, duration and intensity of exercise (Nieman 1994). Furthermore, this study showed a decrease in mitochondrial Bcl-2, which may be due to exercise-induced apoptosis in muscles of mdx mice (Sandri et al., 1997). It is therefore inconceivable that 63% of lymphocytes were apoptotic immediately after exercise as the translocation of phosphatidylserine to the outer cell sheet, which is the basis for the cytometric determination of apoptosis, would invariably require it. A further explanation for the divergent results can be explained by the difference in wells of the lymphocyte populations performed by the different studies and the different methods of analyzing apoptosis. The results of the experiments carried out show a lack of correlation, which can be explained by the different sensitivities of the test for different aspects of cell death. The cause of the DNA damage in the SCGE test is not entirely clear, but oxidative stress is a strong possibility. The percentage of apoptotic and necrotic cells in the flow cytometric results are not as high as expected, although it is possible to extrapolate certain trends among the different test subjects. It is important to obtain reliable and sensitive diagnostic technology for rapid assessment of immune status and associated risk of. susceptibility to disease on the sports field. The results presented here have direct implications for the use of the SCGE assay in studies with human blood cells. This may be beneficial in allowing coaches and athletes to better plan training and competition schedules and in identifying and preventing the so-called "overtrained" athlete who is apparently more sensitive to UR-TIs due to possible immunodepression. Haslett, (1994) Apoptotic neutrophils are phagocytosed by fibroblasts with participation of the fibroblast vitronectin receptor and involvement of a mannose/fucose-specific lectin, Journal of Immunology.
FLOW CYTOMETRY
Apoptotic quantification by detection of phosphotidylserine with annexin V -FITC conjugate
Materials and method
EXERCISE TESTING
Materials and method
